The present invention is directed to modified proteins having at least one point of conjugation with, for example, a drug resulting in specific isomers of the protein-drug conjugate, and to methods for such conjugation resulting in the specific isomers. The invention is further directed to antibodies to which cytotoxic agents and/or cytostatic agents can be conjugated resulting in specific isomers and methods for their conjugation.
Monoclonal antibodies (mAbs) are a valuable weapon in the battle against cancer. mAbs are also used in the treatment of immune disorders. To further advance the use of mAb-based therapies for cancer and immune disorders, a number of novel approaches have been explored. One approach is to increase the cytotoxic potential of mAbs against tumor cells by attaching cell-killing payloads. Molecules such as protein toxins, radionuclides, and anti-cancer drugs have been conjugated to certain mAbs to generate immunotoxins, radioimmunoconjugates, and antibody-drug conjugates (ADCs), respectively.
Factors which have previously been considered in developing ADCs have included the choice of antibody, and optimizing the potency of the drug component, the stability of the linker, and the method by which the drug was covalently attached to the mAb. The common convention for producing ADCs conjugated through the disulfide bonds has been by reducing all inter-chain disulfide bonds of an antibody and reacting all the reduced nab thiols with a compound capable of interaction with all the reduced thiols, forming uniformly-substituted ADCs with 8 drugs/mAb, i.e. “fully loaded,” without the ability to obtain specificity for certain site of conjugation.
For example, the antigen CD30 is highly expressed on cancers such as Hodgkin's disease (HD) and anaplastic large cell lymphomas (ALCL). This expression of CD30, coupled with limited expression on normal cells, makes it an attractive target for ADC therapy. The chimeric mAb directed to CD30, cAC10, has antitumor activity against HD both in vitro and in subcutaneous and disseminated SCID mouse xenograft models. The anti-tumor activity of cAC10 was enhanced by generating fully loaded ADCs in which all eight of the interchain thiols were linked to derivatives of the cytotoxic agent auristatin E as the drug component. These ADCs were highly effective in murine xenograft models at well-tolerated doses.
Because the convention in the production of ADCs has been to fully load them with drug, it was not previously appreciated that partially-loaded ADCs could have the same or greater therapeutic efficacy. Further, methods did not exist which could take into consideration that other substitution patterns on antibodies could produce equal or better therapeutic efficacy with equal or lower toxicity. These and other limitations and problems of the past are solved by the present invention.